Circulating tumor DNA using a plasma-only assay predicts survival in patients with oligometastatic colorectal cancer after definitive therapy

Introduction

The use of liquid biopsy, and more specifically the detection of circulating tumor DNA (ctDNA) in cancer has become a reality in the treatment of patients with cancer (1). Different applications have been suggested for this technology which includes molecular profiling as an alternative to tumor tissue, treatment monitoring and more recently detection of residual disease after definitive therapy and early diagnosis of cancer (2-5). Various applications for this technology have been evaluated for ctDNA, mainly looking into mutations or alterations in ctDNA (6). For instance, tumor-informed approaches where a personalized panel is built based on sequencing on tumor tissue to track in plasma. These assays have demonstrated the ability to detect molecular residual disease (MRD) in different tumor types such as colon, breast, lung and head and neck, among others (7-11). However, this approach is limited due to the need of archival tissue. Tumor naïve approaches, which track specific alterations in plasma that are either common in cancer or tumor-specific, can overcome this limitation (12). Other alternatives include the analysis of epigenetic changes such as methylation which is usually observed in cell free DNA in patients with cancer (13). One tumor naïve approach which combines mutation-based and methylated-based alterations is Guardant Reveal (formerly LUNAR-1). This assay has shown that ctDNA detection in the perioperative setting of localized disease can correlate with recurrence in colorectal cancer (CRC) and breast cancer (14-16).

CRC is one of the tumor types where ctDNA has emerged as a key tool in the use of these assays. For instance, different studies have shown the applicability of these assays in terms of molecular profiling, prognostic stratification and treatment monitoring (17). Moreover, detection of MRD in plasma of ctDNA has become a reality for many patients. Different published studies suggest that ctDNA detection could stratify patients with higher risk of relapse and could be used as a tool to personalize adjuvant therapies (18,19). A recent prospective study has shown that the use of ctDNA to stratify patients to receive adjuvant therapy after resection in stage II colon cancer did not impact outcomes, with many patients avoiding the use of adjuvant chemotherapy (20). However, prospective phase III studies are still underway to evaluate the potential impact in risk of recurrence and survival. In stage IV, the detection of ctDNA after surgery has been associated with recurrence, especially in the case of liver metastases using tumor informed assays (which relies on the sequencing of the resected tumor) (21,22). Although the use of adjuvant chemotherapy is well established after stage II and III CRC, the value of chemotherapy after definitive-intent metastatic treatment (either surgery or radiation) is arguable, especially in terms of survival (23-25). Therefore, ctDNA could also potentially guide the decision of adjuvant therapy in this setting. The impact of ctDNA detection in resected metastases from CRC have not yet been evaluated using a tumor-naïve assay that combines both mutation-based and methylation-based approaches.

In our study, we hypothesized that the detection of ctDNA after locoregional treatment (either surgery or radiation) is associated with a higher risk of recurrence, which could impact survival. To test our hypothesis, we designed a prospective study in patients with CRC with oligometastatic disease where surgery or radiation was delivered with definitive intent. We analyzed plasma samples before and after treatment using a tumor-naïve mutation and methylation-based assay (Guardant Reveal). Our goal was to explore whether ctDNA detection after treatment in oligometastatic disease in CRC could serve as a prognostic biomarker. This study is reported following the REMARK reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-24-819/rc) (26).

Methods

We developed a single center prospective observational study including consecutive patients with oligometastatic CRC treated with definitive intent at the Comprehensive Cancer Centre Clara Campal (Spain). The study received approval from the institutional review board of HM Hospitales (No. 18.12.1344-GHM on 19 March 2019) and was conducted in accordance with the Declaration of Helsinki (as revised in 2013). All patients signed an informed consent form before enrollment.

Patients with stage IV colorectal adenocarcinoma were included in the study. Eligible patients were candidate for: (I) surgery or (II) stereotactic body radiation therapy (SBRT) of all the metastases after discussion in our multidisciplinary tumor board. Patients were included in this study only if the metastatic disease was treated completely with these therapies. Patients were included if they had synchronous tumors, diagnosed simultaneously with the primary tumor, or if they developed metastases during surveillance following the removal of the primary tumor (metachronous). Patients consented for a plasma sample collection before definitive treatment of the metastases. This sample was collected within 2 weeks of the procedure and no earlier than fourteen days from the last dose of systemic therapy. The second plasma sample (follow up) was collected approximately four weeks after surgery or SBRT and before any adjuvant therapy was administered (if applicable). The adjuvant chemotherapy regimens included capecitabine, fluoropyrimidines in combination with oxaliplatin, and, in two cases, the addition of anti-epidermal growth factor receptor (anti-EGFR) and anti-angiogenic therapy. Patients were followed for survival and disease status. Clinical and radiological assessments were performed every 3–4 months for the first two years of follow-up and then every 6 months, as per standard of care in our institution. Main epidemiological, clinical and pathological characteristics were recorded using chart review.

Plasma samples were analyzed using the Guardant Reveal (former LUNAR-1) (Guardant Health, Redwood City, CA, USA) assay, as previously reported (14). Briefly, this tumor naïve assay uses next generation sequencing approach to detect multiple somatic alterations, including single nucleotide variants, fusions and copy number alterations as well as epigenomic alterations such as methylation, compared to normal DNA. Genomic alterations are filtered to remove variants of likely benign origin (such as clonal hematopoiesis of indeterminant potential). These alterations have been defined based on their frequency in CRC, and there is no knowledge of the specific molecular alterations in the patient’s tumor. In our study, plasma samples were stored at our institution and then sent for ctDNA extraction and analysis. The analysis of ctDNA was performed blinded to clinical outcomes. Results were not provided to patient or clinician in real time, and clinical decisions were not made based on these results.

Statistical analysis

A preliminary descriptive analysis of the patients’ characteristics was conducted by calculating central tendency and dispersion measures of quantitative variables. For qualitative variables, comparison of proportions was tested by using the χ² test or the Fisher exact test, whenever necessary. The primary endpoint of the study was to assess the correlation of ctDNA detection after definitive therapy of the oligometastatic disease with disease-free survival (DFS). We also analyzed the correlation of ctDNA detection at this timepoint with overall survival (OS). We analyzed the correlation of pre-treatment ctDNA detection with OS and DFS as well as the changes in ctDNA after definitive therapy. Both DFS and OS were estimated from the time of the SBRT or surgery to recurrence or death (DFS) or death (OS) using the Kaplan-Meier method. Estimations of DFS at 1 year and OS at 2 years for each group were calculated. To determine hazards ratios, a Cox proportional regression model was used to assess the relationship between mortality and/or recurrence and the presence of ctDNA after surgery or SBRT. A P value of less than 0.05 was considered to indicate statistical significance in all analyses. Data management, statistical calculations, and graphical plots were conducted using the R statistical software.

Results

A total of 25 consecutive patients were included in the study from April 2019 to September 2021; one patient was excluded due to histology not being adenocarcinoma. Furthermore, baseline and follow up plasma samples from two patients were not evaluable: one patient was lost to follow-up, and the plasma samples from another patient failed enrichment. Three pre-treatment and post-treatment samples were also deemed non-evaluable. Main reasons were failed enrichment (N=2), failed methylation portioning (N=2), low coverage (N=1) and contamination (N=1). Therefore, a total of 19 patients were evaluable for ctDNA at baseline, 19 patients at follow up and 16 patients at both timepoints (Figure 1).

Figure 1 Flow diagram of the samples and patients in the study. This figure summarizes the number of patients enrolled in the study, the number of patients who were evaluable in the study, the number of patients with evaluable plasma samples at baseline, at follow-up and the number of patients who were evaluable for both. ADC, adenocarcinoma; ctDNA, circulating tumor DNA; FU, follow-up.

Main characteristics of the evaluable population at baseline are summarized in Table 1. Briefly, most patients were males (62.5%), median age was 66 years with primary site of metastases in the liver (12 patients, 50%). Seven patients (29%) have more than one metastatic lesion but were treated in the same procedure with definitive intent. As for the definitive therapy, 13 patients had SBRT (54.2%) while the remaining 11 had surgery. All except for one patient had progressed or died at the time of the analysis with a median follow up from the second plasma sample of 20.7 months. Nine patients have died while other fourteen patients have progressed with multiple sites of metastases as the most frequent pattern of recurrence (8/23, 34.8%).

Baseline detection of ctDNA and correlation with outcomes

At baseline, ctDNA was evaluable in 19 patients: ctDNA was detected in 12 of them (63%). The median time between plasma sample and SBRT or surgery was 15 days (8–36 days). There was no correlation of ctDNA detection with stage at diagnosis, RAS mutation, number of metastases, synchronous/metachronous disease, or location of metastases as summarized in Table 2.

A total of eight patients were deceased, ten had recurrence but still alive, and one was free of disease and alive at the time of analysis. Baseline ctDNA detection was not associated with OS [hazard ratio (HR): 1.94; 95% confidence interval (CI): 0.38–9.86; P=0.41] (Figure 2A). OS at 2 years was 75% (54–100%) in ctDNA positive and 71% (45–100%) in ctDNA negative. Baseline ctDNA detection did not predict either DFS (HR: 1.15; 95% CI: 0.43–3.07; P=0.78) (Figure 2B): DFS at 1 year was 29% (95% CI: 8.9–92) in ctDNA negative and 8.3% (95% CI: 1.3–54%) in ctDNA positive.

Figure 2 Outcomes according to detection of ctDNA at baseline. There were no statistically significant differences at baseline between patients with detection and no detection groups in overall survival (A) and disease-free survival (B). ctDNA, circulating tumor DNA.

Post-treatment detection of ctDNA and correlation with outcomes

In contrast, ctDNA after surgery or SBRT was evaluable in 19 patients, of whom 16 were also evaluable at baseline; detection was observed in 7 of the 16 patients (44%). Among these 7 patients, five relapsed and died (71.4%) and other two relapsed but are still alive on follow up. Of the twelve patients without post-treatment ctDNA detection, ten patients relapsed but are still alive (83.3%), one patient has died and other patient is alive free of disease. There were no significant differences between patients with detectable and undetectable ctDNA in terms of stage at diagnosis, RAS status, number of metastases, site of metastases and type of treatment between patients with detectable and undetectable ctDNA post-treatment, as seen in Table 3. The median time between SBRT or surgery to plasma sample collection was 34 days [slightly longer for patients with ctDNA detection (47 days) compared to those without (33.5 days) (P=0.15)].

We analyzed the impact of post-treatment ctDNA on OS and DFS. The 2-year OS for the whole post-treatment cohort (N=19) was 75% (95% CI: 58–97%). Patients without ctDNA detection had a higher probability of being alive at 2 years, 92% (95% CI: 77–100%), compared to patients with ctDNA detection post-treatment, 57% (95% CI: 30–100%) (Figure 3A). Overall, detection of ctDNA post-treatment, was associated with worse survival (HR: 11.28; 95% CI: 1.31–97.05, P=0.03). We also correlated ctDNA detection post-treatment with DFS. All patients with ctDNA detection progressed or died within the first year of follow up while the 1-year DFS rate for patients with undetectable ctDNA was 33% (95% CI: 15–74%) (Figure 3B). We further quantified the effect size for no detection of ctDNA after treatment and we found a non-significant trend toward longer DFS (HR: 2.97; 95% CI: 0.97–9.06, P=0.05).

Figure 3 Outcomes according to detection of ctDNA at follow-up (4 weeks after definitive surgery or SBRT). Patients with ctDNA detection during follow-up had shorter overall survival compared to patients with no detection (A). A similar non-statistically significant trend is observed in DFS (B). ctDNA, circulating tumor DNA; DFS, disease-free survival; SBRT, stereotactic body radiation therapy.

We analyzed the impact of post-treatment ctDNA detection according to the site of metastasis, although our results were not statistically significant, likely due to the small sample size. In patients with liver metastases (N=10), OS at 2 years was 57% in the undetected group versus 33% in the detected group (P=0.13), while 1-year DFS was 29% in the undetected ctDNA group versus 0% in the detected ctDNA group. In patients with lung metastases (N=3), OS at 2 years was 100% in both groups, while 1-year DFS was more frequent in the non-detected group (50% vs. 0%, P=0.81). In patients with more than one site of metastasis (N=6), OS at 2 years was similar in both groups (33%), while 1-year DFS was 33% in the negative ctDNA group and 0% in the detected group (P=0.27). We also analyzed the impact of the treatment modality on the detection of post-treatment ctDNA and the time to recurrence or death. In patients who received surgery as definitive treatment (N=10), all patients progressed within the first year in both groups (positive and negative ctDNA), though there was a trend toward a higher number of patients alive at 2 years in the undetected ctDNA group (OS at 2 years: 29% vs. 0%, P=0.46). In patients who received radiation as definitive treatment (N=9), both the 1-year DFS and 2-year OS rates were higher in patients with undetected ctDNA: 1-year DFS 80% vs. 0% (P=0.27) and 2-year OS 100% vs. 50% (P=0.46).

ctDNA kinetics and correlation with outcomes

We also analyzed the impact of the change in ctDNA in those patients were both samples were available (N=16). Patients were classified into four groups according to ctDNA detection (pre/post): persistently positive (+/+) (N=4), clearance (+/−) (N=6), positive converters (−/+) (N=2) and persistently negative (−/−) (N=4). These groups were significantly associated with OS and DFS (P=0.04). Overall, persistently negative patients have the best survival with all patients alive at the time of the cut-off. Patients who cleared ctDNA also had a high 2-year OS (83%, 95% CI: 58–100%). In contrast, patients with persistently positive ctDNA or positive converters had the worst 2-year OS: 50% (95% CI: 19–100%) and 50% (95% CI: 13–100%), respectively (Figure 4A). Regarding DFS, similar results were observed as shown in Figure 4B. One-year DFS was 50% (95% CI: 19–100%) in patients with persistently negative ctDNA and 17% (95% CI: 2.8–100%) in patients with ctDNA clearance. All patients with persistently positive ctDNA or ctDNA conversion had progressed or died one year after the treatment. These findings confirm that detection of ctDNA after definitive therapy is more relevant than baseline detection.

Figure 4 Outcomes according to changes in detection of ctDNA from baseline to follow-up. OS (A) and DFS (B) based on the changes in ctDNA between baseline and follow-up. Group 1: persistently positive; Group 2: clearance; Group 3: positive converters; Group 4: persistently negative. Patients who are persistently negative have better outcomes than the rest of the groups while patients who become positive after treatment have worse outcomes. ctDNA, circulating tumor DNA; DFS, disease-free survival; OS, overall survival.

Discussion

We have demonstrated in this prospective study that ctDNA can be detected using a tumor naïve assay (Guardant Reveal/LUNAR-1) in patients with oligometastatic CRC both before and after surgery or SBRT. We found that the detection of ctDNA post-treatment is associated with shorter OS. Moreover, there is a non-statistically significant trend toward shorter DFS. In our cohort, these differences were not observed based on ctDNA status prior to treatment. We also observed that those patients with persistently negative ctDNA had the best survival while those who were persistently positive or converters had worse outcomes. Therefore, the detection of ctDNA post-treatment appears to be critical to evaluate the potential risk of recurrence and death in patients with oligometastatic disease treated with definitive intent.

The use of ctDNA to detect MRD in CRC has been extensively investigated, particularly in localized, curative setting. Different independent studies and a large meta-analysis have shown that the detection of ctDNA after surgery and/or adjuvant chemotherapy is associated with recurrence and shorter survival mainly in patients with localized disease (21). In patients with resected metastases, a study including 230 patients using a tumor informed approach evaluating ctDNA four weeks after treatment showed a clinically meaningful and significant association of the detection of ctDNA with progression-free survival (HR: 5.9), though the association with survival has not yet been reported (27). In our study, a trend to worse DFS in patients with ctDNA detection after treatment was also observed, but may not be statistically significant due to sample size. A study involving 76 patients with liver only colorectal metastases showed that ctDNA detection after therapy was associated with shorter DFS (HR: 2.09) and a trend to shorter OS (HR: 1.65) (22). However, this study was limited only to liver metastases (while ours included other sites of metastases) and used a droplet digital polymerase chain reaction (PCR) targeting limited genes found in the primary tumor sequencing. More recently, a study involving 87 patients with oligometastatic disease showed that ctDNA detection after intervention was associated with prolonged DFS (HR: 2.68). This study used a tumor-informed assay and as the ctDNA testing was performed after adjuvant therapy (which was delivered in more than half of the patients) (28). In contrast, our study used a tumor-naïve approach for ctDNA analysis and the plasma samples were collected before any adjuvant therapy. This is one of the first studies showing the value of this assay which evaluates both genomic and epigenomic signals in the oligometastatic setting treated with curative intent, which is not a common scenario in CRC.

In our study, as previously explained, we used a tumor-naïve ctDNA approach which analyzes both genomic and epigenomic alterations in plasma. This assay has been previously tested in CRC in stage I-IV (14). In this study, ctDNA was analyzed one month after completion of definitive therapy. However, more than half of the patients had that sample collected after completion of adjuvant chemotherapy. Nevertheless, ctDNA detection was significantly associated with recurrence (HR: 11.2) regardless stage or use of adjuvant therapy. In this study, there were a total of sixteen patients with metastatic disease and the impact of ctDNA detection on worse recurrence/progression free survival was maintained (HR: 6) and some of these patients received adjuvant therapy, as seen in the supplementary material of this work. However, the site of metastases and the definitive treatment (surgery or radiation) was not provided and therefore a head-to-head comparison is limited. Our assay is specific for cancer patients and we removed variants of likely benign origin such as clonal hematopoiesis of indeterminate potential, that comes from the aging cell. We did not evaluate our assay in healthy donors; however previous studies using same assay did not show detection in patients without cancer (29). Our study shows a detection of 40% after definitive intent therapy, which is in line with some of the other abovementioned study, using tumor-informed approach. This rate is significantly higher than the detection rate of our assay in stage II CRC as recently showed in the COBRA study (30). Thus, our assay may be as effective as others in detecting ctDNA in stage IV oligometastatic CRC, with the added benefit of a potentially reduced turnaround time and cost, (as it does not require tumor tissue availability and sequencing). In short, our study adds further evidence to the literature of the performance of this assay as an option to detect ctDNA in CRC.

This study has several limitations. First, the sample size is small, with only sixteen patients evaluated at both timepoints and ctDNA not evaluable in few cases due to processing issues. This limitation precludes the study from drawing definitive conclusions and from conducting potential multivariate analysis to adjust for prognostic factors, underscoring the need for confirmatory studies. Second, despite all patients being treated with a radical intent, there is significant heterogeneity in the modalities and extent of disease among the patients. We did not compare our approach directly with other sensitive methods, such as droplet digital PCR or tumor-informed ctDNA assays. Lastly although the study was prospective, the results were not analyzed in real time and therefore information was not readily available for clinicians to make decisions. Ongoing studies (such as COSMOS-CRC-03 and AURORA) using this and other assays are evaluating whether the detection of ctDNA could ultimately help on intensifying therapy and improve outcomes in patients with oligometastatic disease (31).

Conclusions

In conclusion, our study provides further evidence in CRC of the role of ctDNA as a predictive biomarker of survival and recurrence, that warrants further validation in larger cohorts. From a clinical perspective, our results suggest that patients with detectable ctDNA after surgery and radiation may experience shorter survival and earlier recurrence. Therefore, based on our results, we hypothesize that this assay could serve as a valuable tool for clinicians to guide the intensification or de-intensification of treatment following surgery or radiation therapy. This could involve adding more intensive chemotherapy (as proposed in the AURORA study) or increasing radiological surveillance if ctDNA is detected. In contrast, patients without detectable ctDNA might be spared from adjuvant therapies (as proposed in the ongoing COSMOS-CR-03 study). There is a need to pursue prospective and multicenter studies that goes beyond detection of ctDNA and looks to potential strategies to intercept ctDNA after treatment of oligometastatic disease in CRC.

Acknowledgments

We would like to acknowledge all patients who participated in this study as well as the HM Hospitales Research Institute for their support.

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-24-819/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-24-819/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-24-819/prf

Funding: This research was funded by INTHEOS foundation and the APC was funded by HM Hospitales Foundation.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-24-819/coif). E.S.G. receives consulting fees from GSK. His current institution also receives payments for research from GSK and Rgnta therapeutics. P.P. receives speaker honorarium from Merck and travel grants from Merck and Roche. C.M. receives funding for his institution from Servier. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study received approval from the institutional review board of HM Hospitales (No. 18.12.1344-GHM on 19 March 2019) and was conducted in accordance with the Declaration of Helsinki (as revised in 2013). All patients signed an informed consent form before enrollment.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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